Histomonas meleagridis is notorious for being the causative agent of histomonosis, which can cause high mortality in turkeys and substantial production losses in chickens. The complete absence of commercially available curative strategies against the disease renders the devising of novel approaches a necessity. A fundamental step toward this objective is to understand the flagellate's virulence and attenuation mechanisms. For this purpose we have previously conducted a comparative proteomic analysis of an in vitro cultivated virulent and attenuated histomonad parasite using two-dimensional electrophoresis and MALDI-TOF/TOF. The current work aimed to substantially extend the knowledge of the flagellate's proteome by applying 2D-DIGE and sequential window acquisition of all theoretical mass spectra (SWATH) MS as tools on the two well-defined strains. In the gel-based experiments, 49 identified protein spots were found to be differentially expressed, of which 37 belonged to the in vitro cultivated virulent strain and 12 to the attenuated one. The most frequently identified proteins in the virulent strain take part in cytoskeleton formation, carbohydrate metabolism and adaptation to stress. However, post-translationally modified or truncated ubiquitous cellular proteins such as actin and GAPDH were identified as upregulated in multiple gel positions. This indicated their contribution to processes not related to cytoskeleton and carbohydrate metabolism, such as fibronectin or plasminogen binding. Proteins involved in cell division and cytoskeleton organization were frequently observed in the attenuated strain. The findings of the gel-based studies were supplemented by the gel-free SWATH MS analysis, which identified and quantified 42 significantly differentially regulated proteins. In this case proteins with peptidase activity, metabolic proteins and actin-regulating proteins were the most frequent findings in the virulent strain, while proteins involved in hydrogenosomal carbohydrate metabolism dominated the results in the attenuated one.The unicellular protozoan  Histomonas meleagridis, first classified in 1920 : 5 × 108 bacterial cells/ml), which was grown overnight at 37°C, was added to the cultures. Subsequently, the virulent low-passaged parasites were harvested at passage number 25 and the attenuated high-passaged parasites at passage number 303. A purification protocol consisting of consecutive washing and centrifugation steps was used to collect H. meleagridis cells and to remove most of the bacteria as previously described  and the second biological replicate on the day 2 (sample b), and as a result four protein samples were included in each of the two 2D-DIGE experiments: 25a, 25b, 303a, 303b , G-Dye200 (G200), and G-Dye300 (G300) lysine fluorescent dyes, included in the Refraction-2D™ kit  were reconstituted and used for the minimal labeling of protein samples according to manufacturer's instructions. Each 2D-DIGE experiment consisted of four 2-DE gels. In each gel, three samples were visualized, namely 25a or 25b, 303a or 303b and the IS  were rehydrated for 16–17 h at room temperature in rehydration buffer  to which 0.4% (w/v) DTT and 0.5% (v/v) SERVALYT™ 3–10 Carrier Ampholytes  were added  containing 2% (w/v) DTT for 15 min at room temperature. Then the IPG strips were alkylated under the same conditions and in the same equilibration buffer which contained 2.5% (w/v) iodoacetamide (IAA) instead of DTT.TM Multicolor Broad Range Protein Ladder, 10–260 kDa, Thermo Scientific™, Fisher Scientific (Austria) GmbH, Vienna, Austria] was applied for approximate estimation of protein molecular mass (Mr).For the second dimension, vertical slab gels  were casted simultaneously using the PROTEAN® II multi-gel casting chamber (Bio-Rad®) according to manufacturer's instructions. Subsequently, the equilibrated IPG strips were placed on top of a polymerized stacking gel (18.3 cm × 3 cm × 1.5 mm) using 0.5% pre-warmed agarose, containing 0.05% of bromophenol blue dye, which was used for tracking purposes. The second dimension was carried out using Protean® II xi cell (Bio-Rad®) with a current of 15 mA/gel at 4°C until the tracking dye ran off the bottom of the gels. Fifteen microliters of molecular marker  and 1 μl of the resulting mixture was spotted on a ground steel MALDI target plate  and allowed to dry at room temperature. Spectra data were acquired in MS and MS/MS modes by performing MALDI-TOF/TOF MS . FlexAnalysis 3.0 and Biotools 3.2 software  were used for spectra processing and peak annotation. Processed spectra were submitted to an in-house Mascot server  using the ProteinScape 2.1 software . Subsequent searches were performed in an amino acid database of Five and three protein spots from the 2D-DIGE-α and 2D-DIGE-β experiment, respectively, could not be identified by MALDI-TOF/TOF MS and were analyzed by LC-MS/MS. For this, proteolytically generated peptides were separated by a high-performance chromatography system  and analyzed with a high-resolution hybrid triple quadrupole time-of-flight mass spectrometer  connected via nano-electrospray ionization (ESI) interface.2O with 0.05% TFA was used for loading and de-salting of peptide samples with a flow rate of 5 μl/min. For peptide separation a 25 cm Acclaim PepMap C18 column  was used operated at a flow rate of 300 nl/min. The gradient started with 4% B (80% ACN with 0.1% formic acid) and increased to 35% B in 60 min. It was followed by a washing step with 90% B. Mobile Phase A consisted of ultra-pure H2O with 0.1% formic acid. Mass spectra of HPLC-separated peptides were acquired using an information dependent data acquisition mode (IDA). MS1 spectra were collected in the range of 400–1,500 m/z for 250 ms. The 25 most intense precursors with charge state 2–4, which exceeded 100 counts per second, were selected for fragmentation. MS2 spectra were acquired in the range of 100–1,800 m/z for 110 ms. Precursor ions were dynamically excluded from reselection for 10 s. The nano-HPLC system was operated by Chromeleon 6.8 using DCMS-Link (Dionex) and the MS by Analyst Software 1.6 (Sciex).Remainders of the de-salted peptides after in-gel digest were evaporated and reconstituted in 0.1% aqueous TFA for injection into LC-MS. Sample pre-concentration and de-salting was accomplished with a 5 mm Acclaim PepMap μ-Precolumn  (Dionex). As a mobile phase 2% ACN in ultra-pure HE. coli and H. meleagridis as described above in: Identification of differentially abundant protein spots by MALDI-TOF/TOF. Mass tolerance in MS mode was set with 0.05 and 0.1 Da in MSMS mode for the rapid recalibration search, and 0.0011 Da in MS and 0.01 Da in MSMS mode for the final search. Parameters for database search: trypsin digestion, cysteine alkylation set to iodoacetamide, search effort set to rapid ID. FDR was performed using the integrated tools in ProteinPilot. Global FDR was set to < 1% on protein as well as on peptide level. Following the generally applied so-called “two-peptide rule” only proteins with at least two peptides were further considered as identified  in the combined database of H. meleagridis proteins by LC-MS/MS without previous gel electrophoretic separation, protein digestion was performed using a two-step in-solution digestion protocol with a combination of LysC and trypsin according to the manufacturer's recommendationFor identification of the LC parameters for data independent SWATH quantification analyses were the same as described above for IDA injections. MS parameters were adapted for SWATH. MS1 spectra were collected in the range of 400–1,500 m/z with an accumulation time of 50 ms. Product ion spectra were collected in 34 windows in the range of 400–1,250 m/z with a fixed width of 25 Da. For each window ions were accumulated for 80 ms.IDA identification results (see section Shotgun Protein Identification Using LC-MS/MS) were used to create the SWATH ion library with SWATH Acquisition MicroApp 2.0 in PeakView 2.2 . Peptides were chosen based on a FDR rate < 1%, excluding shared and modified peptides. Up to 6 peptides per protein and up to 6 transitions per peptide were used. Calculation of peak areas of SWATH samples was performed with MarkerView 1.2.1  after retention time alignment and normalization using total area sums.Further statistical evaluations were based on the normalized peak areas. Two different software tools were used: R programming language . As MarkerView does not differentiate between technical and biological replicates an additional statistical evaluation with R was performed.After principal component analysis (PCA), groups were defined and a cRAP proteins and proteins quantified with just one peptide were removed from the MarkerView normalized raw protein list before further processing. Raw peak areas were log2-transformed to approach a normal distribution. On a logarithmic scale, technical replicates were aggregated by arithmetic mean before application of statistical tests.t-Test for independent samples for each protein. To adjust for multiple testing, the method of Benjamini and Hochberg was used to control the FDR .Differential expression of proteins was assessed using two-tailed H. meleagridis-specific proteins and their proposed function. The identified proteins were found to be significantly differentially expressed between the two strains by the 2D-DIGE-α, 2D-DIGE-β, and SWATH MS experiments. The proposed functions were attributed to identified proteins based on their Gene Ontology terms, InterPro entries and existing literature. The fold upregulation values, which were acquired from Delta2D software version 4.7 (Decodon GmbH) and SWATH MS quantification measurements, were mapped to certain colors. In this way, the H. meleagridis-specific proteins, which were represented by source nodes, were visually characterized and their level of expression was indicated.The open source software platform Cytoscape version 3.5.1 . The landmark spot, which was distinctive at pI 5 and a Mr of ~50 kDa , was known to be actin of H. meleagridis origin based upon a previous study  were more abundant in the attenuated strain  were more numerous in the protein samples of the virulent strain  overexpressed by the 2D-DIGE-α and 2D-DIGE-β analysis, respectively, matched with entries from the aforementioned databases   Tables , while i) Tables . Between) Tables . In the ) Tables , while i) Tables . In the ) Tables .H. meleagridis-specific proteins   Figure . Six pro) Figure . AlthougpI Table . The act Figures . In the  Figures . Additio Figures . The remMr of ~110 kDa  and attenuated (303a and 303b) re Table .The fold change in upregulation values ranged between 2.0 and 17.8 for proteins of the virulent strain and between 2.0 and 3.4 for proteins of the attenuated one Table , 4C. TheThe 20 overexpressed proteins in samples of the attenuated culture represented 15 different proteins, out of which only coronin was shown to be also significantly upregulated in the gel-based experiments Table . The uprE. coli DH5α. However, an average number of 151 and 158 proteins were identified to be of bacterial origin in protein samples obtained from the in vitro cultivated virulent (25a and 25b) and attenuated (303a and 303b) strains, respectively.In contrast to the gel-based experiments, none of the above 42 significantly differentially expressed proteins belonged to H. meleagridis cultures -, plasminogen (PLG)- and collagen-binding protein with localization on T. vaginalis surface , namely surfactant protein B-like (SPB-like), being overexpressed in the virulent strain. SPB contains a motif of six cysteine periodically located residues, which is commonly found in proteins of the saposin-like family -containing protein. This motif is found in enzymes that, in addition to chitin-binding, also degrade bacterial cell walls  and one protein (ADK protein) that acts in the hydrogenosome were shown to be overexpressed in the virulent strain by the gel-based and gel-free experiments. Except from the single GAPDH spot and ADH, the detected overexpression of two other glycolytic proteins , as well as six hydrogenosomal enzymes  was the exclusive property of the attenuated parasite.T. vaginalis and H. meleagridis, carbohydrate metabolism is fermentative. The part of the glycolytic pathway that takes place in the cytoplasm results in pyruvate or malate production, which are further converted to hydrogen, carbon dioxide and acetate in double-membrane organelles called hydrogenosomes , -2 (64 kDa), and -3 , which is an important oxidative stress defense in T. vaginalis, was only reported in the attenuated one (Ellis et al., in vitro fluctuating oxygen levels. Alternatively, the fact that the overexpressed iron hydrogenases were mapped to different contigs can itself constitute a coping mechanism that protects hydrogenosomal metabolism from oxygen and might explain the higher adaptation of this strain to the in vitro environment.In amitochondriate anaerobic protozoa, such as H. meleagridis proteomic analysis that utilized conventional 2-DE (Monoyios et al., in vitro conditions in comparison to the high-passaged attenuated strain. This was manifested by the upregulation of proteins related with stress response in the former and the overexpression of cell division proteins in the latter. Additionally, the upregulation of different components of the carbohydrate metabolism between these strains might further support this view. Molecules that are part of the virulence arsenal of other related parasites such as CPs and PFPs were detected in the virulent strain, a feature in large part identified by the SWATH MS experiment. Finally, both gel-based experiments identified multiple modifications of ubiquitous proteins such as actin and GAPDH in the virulent strain, which might indicate alternative functions and localizations. This finding was supported by the conventional 2-DE comparative study and should be addressed in greater detail by future investigations.This study corroborated, and to a greater extent supplemented the findings of the first The mass spectrometry proteomic data were deposited to the ProteomeXchange Consortium via the PRIDE (Vizcaíno et al., Any other data supporting the conclusions of this manuscript can be made available by the authors, without undue reservation, to any qualified researcher upon request.IB, AM, and MH contributed conception and design of the study. MP assisted with 2D-Gels. KN, KH, and SS performed mass spectrometry analysis and wrote some material sections of the manuscript. AM, KN, and KH organized the database and performed the statistical analysis. AM and IB wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rhizopus sp. was completely inhibited. By using the new medium along with the DV-AM method, we succeeded in the isolation of aflatoxigenic fungi from non-agricultural fields in Fukui city, Japan. The fungi were identified as Aspergillus nomius based on their calmodulin gene sequences. These results indicate that the new medium will be useful in practice for the detection of aflatoxigenic fungi in soil samples including those from non-agricultural environments.The dichlorvos-ammonia (DV-AM) method is a simple but sensitive visual method for detecting aflatoxigenic fungi. Here we sought to develop a selective medium that is appropriate for the growth of aflatoxigenic fungi among soil mycoflora. We examined the effects of different concentrations of carbon sources (sucrose and glucose) and detergents (deoxycholate (DOC), Triton X-100, and Tween 80) on microorganisms in soils, using agar medium supplemented with chloramphenicol. The results demonstrated that 5–10% sucrose concentrations and 0.1–0.15% DOC concentrations were appropriate for the selective detection of aflatoxigenic fungi in soil. We also identified the optimal constituents of the medium on which the normal rapid growth of  Aflatoxins (AFs) are highly toxic, carcinogenic, and teratogenic secondary metabolites produced by fungi . The conAspergillus flavus Link and Aspergillus parasiticus Speare [AFs are produced mainly by certain strains of s Speare . These fs Speare ,4,5. Afls Speare . These fs Speare .Aspergillus group have been reported to produce AFs: A. nomius [A. pseudotamarii [A. bombycis [A. parvisclerotigenus [A. minisclerotigenes [Several other species of the . nomius ,8,9, A. otamarii ,11, A. bbombycis , A. parvotigenus ,14,15, Aotigenes , and morAspergillus section Flavi using Aspergillus flavus and parasiticus agar (AFPA) medium [Aspergillus section Flavi, the ability of the fungi to produce aflatoxins need to be confirmed by another analytical method noted in method (2). These methods are useful and fruitful techniques but are time-consuming and often require some special facilities, especially in step (2). For more practical use, a single-step method for the selective detection of aflatoxigenic fungi from environmental samples is desirable.Many researchers have reported the isolation of aflatoxigenic fungi from the soil in agricultural fields , and the researchers usually used multiple procedures: (1) the isolation of ) medium , modifie) medium , or coco) medium ; (2) the) medium ,20,21,22) medium ,26,27,28) medium ,30,31, aWe previously developed a sensitive and simple visual detection method, the dichlorvos-ammonia (DV-AM) method , in whicSince these precursors contain anthraquinone moieties in their structures, the mycelial colors are markedly changed from the original yellowish colors to purple-red through the pH change by ammonia vapor. Monitoring this color change makes it easy to identify aflatoxigenic fungi, as the colors of non-aflatoxigenic fungi do not change. We demonstrated that with the DV-AM method, aflatoxigenic fungi were isolated from sorghum fields in Japan , and thaIn our previous work, we routinely used GYD ) medium for the screening of aflatoxigenic fungi ,35, and Aspergillus section Flavi. Using this medium, we isolated aflatoxigenic fungi from a non-agricultural field in Fukui city, Japan. This is the first report that aflatoxigenic fungi are present in the Hokuriku area in Japan. We will report the distribution of aflatoxigenic fungi in Japan in another paper.In the present study, we examined the effects of carbon sources and detergents as well as their concentrations to optimize media for the DV-AM method. We were able to establish ‘recipes’ of the optimal medium constituents for aflatoxigenic fungi as well as A. parasiticus strain 95DM accumulates norsolorinic acid (NA), which is an AF precursor containing anthraquinone moiety in its molecule [Since the molecule , the colDuring this study, we discovered that a FUT flowerbed soil endogenously contained some aflatoxigenic fungi. To examine the competency of media for the sensitive detection of aflatoxigenic fungi, the same FUT soil (approx. 30 mg) was cultured on GY(2-0.5)-0.05% DOC-CP-DV or YES(0.5-10)-0.05% DOC-CP-DV medium B to whicTo determine which carbon source (sucrose or glucose) is appropriate for the DV-AM method, we analyzed the FUT soil using either YES(2-10)-0.05% DOC-CP C, left oTo determine the appropriate concentration of the carbon source, we examined the same soil using the same medium except for the varied concentration (0–20%) of sucrose . A sucroWe examined the effect of detergents , and Tween 80 (TW-80)) on the growth of aflatoxigenic fungi and other microorganisms in FUT soil . When thA. parasiticus SYS-4, and A. parasiticus NIAH-26 were cultured on the same plates containing various DOC concentrations for 3 days, each fungal size changed with the increase in the DOC concentration . These results indicated that A. nomius is more tolerant to DOC than A. parasiticus, and that a DOC concentration >0.15% may be more appropriate for the detection of the A. nomius strain.When three strains, i.e., FUT-A1 (an isolate from a FUT flowerbed soil), ntration A, lower.We performed the same experiments using TX-100, which has also been used to make colonies compact . At 0.03When the same experiments were done using TW-80, no aflatoxigenic fungi were detected at all concentrations up to 1% TW-80 C, lower.Rhizopus sp. fungi usually disturbs the observation of other microorganisms in an agar plate, because Rhizopus sp. grows rapidly and spreads throughout the petri dish surface within 1 day. When we inoculated a Rhizopus sp. strain onto the center of agar plates containing various concentrations of DOC, the mycelial density significantly decreased at 0.01% DOC, and fungal growth was greatly inhibited at 0.03% DOC. DOC concentrations >0.05% completely inhibited the growth of Rhizopus sp.  in which no aflatoxigenic fungi had been detected. When 15 mg of soil A and 15 mg of soil B was added to SYS-4 spores and analyzed by the DV-AM method, approx. 68% and 83% of aflatoxigenic fungi were recovered from soil A and soil B, respectively. When twice that amount of either soil was used, the recovery of the aflatoxigenic fungi decreased: ≥34% of aflatoxigenic fungi could be recovered C. SimilaFor our evaluation of the optimal soil storage conditions, an aliquot of the same FUT soil was treated by freeze-thawing. One to three times freeze-thawing did not significantly affect the viabilities of the fungi, and the results obtained with the soils and the soil suspensions were almost the same (data not shown).Aspergillus including aflatoxigenic fungi appeared on this agar medium as colonies with white fluffs from the surface; in contrast, flat yellow colonies showing radiation-like wrinkles were observed underneath the plates (We attempted to isolate aflatoxigenic fungi from three sites in Fukui city using the YES(2-10)-0.1% DOC-CP medium and the DV-AM method. The soil from the flowerbed, which was from the same site from which FUT-A1 was isolated, showed brilliant red colonies A. Soils e plates D. Based 1 and AFG1) were observed -0.1% DOC-CP agar medium together with the agar part underneath the colony was extracted and analyzed by TLC, VHA and aflatoxins  of DOC to the medium was useful to more selectively detect the aflatoxigenic fungi than those of other microorganisms. Also, colonies of Aspergillus section Fravi fungi including aflatoxigenic fungi showed characteristic morphology: Flat colonies having radiation-like wrinkles from the underside. Confirmation of the morphological characters and the color change from pale yellow to purple red by AM treatment can provide more promising determination of aflatoxigenic fungi.This work indicated that the YES(2-10)-0.1% DOC-CP medium is appropriate for selective detection of aflatoxigenic fungi in soils. High concentration (5–10%) of sucrose was appropriate to make brilliant purple-red colonies among many colonies of other microorganisms on the medium. Also, since Rhizopus.TX-100 has also been commonly used to make fungal colonies compact, whereas concentration dependency of TX-100 on mycelial sizes were different from that of DOC. Since low concentration of TX-100 drastically decreased the sizes of aflatoxigenic fungi as well as other microorganisms, discrimination of the aflatoxigenic fungi from others were not easy. Interestingly, TW-80 did not affect the growth of the aflatoxigenic fungi as well as other microorganisms including fungi belonging to genus The mechanisms of growth inhibition by detergents are not known. It is generally accepted that a detergent molecule or micelle binds to cell membranes of microorganisms and it affect certain functions of cells . For exaA. nomius strain was more tolerant than A. parasiticus, and we also found that A. flavus showed similar tolerance to DOC to A. parasiticus. Since Massi et al. reported primer sequences of PCR analysis for selective detection of A. nomius strains [A. nomius strains from environmental samples such as soils.This work also showed that  strains , combinaRhizopus sp. and expected to be yeasts, Penicillium, and so on. We often observed some pale purple colonies on the selection medium and their colors changed to more intense purple-red by AM treatment. These colonies were smaller than those of aflatoxigenic fungi and we confirmed that these fungi did not produce any aflatoxins by TLC analyses (data not shown). These color metabolites affected with AM treatment seems to be important to study soil microorganisms.In this work, we did not investigate other microorganisms in soils, which were This work thus showed that microorganisms in soils inhibited growth of the aflatoxigenic fungi. Although 10-fold dilution of soil did not affect the inhibition, combination of several microorganisms may be useful for inhibition of the fungal growth in crops. Soils might be able to regulate infection of aflatoxigenic fungi in crops.A. flavus and A. parasiticus do not readily move downward beyond the upper 6 cm of soil surface, when those conidia were applied to the soil surface [This work clearly showed that most of all the aflatoxigenic fungi were present as condiospores in the soils. Also, the soils were obtained from non-agricultural places such as FUT campus and a city park. Since crops heavily infected with the aflatoxigenic fungi has not been reported in Japan except at Miyazaki University, the reason the conidiospores are present in the non-agricultural fields remains to be studied. We found that the aflatoxigenic fungi were present at up to 8 cm depth from the surface of the soil. Horn et al. reported that conidia of  surface , probablA. nomius. Although the aflatoxigenic fungi have been isolated from agricultural fields in Okinawa area [Aflatoxigenic fungi have been thought to be rare in Japan, especially in the area of the north of the central part in Japan . Howeverawa area , Kyusyu awa area , Kanto aawa area ,48, and awa area ,48,49. TThese results suggested that the DV-AM method using the improved medium will be a promising method for clarification of distribution and movement of the fungi in various environments. In fact, we have already started screening of aflatoxigenic fungi in many places and various environments in Japan. We will report those results in another paper soon.In this study, we demonstrated that addition of appropriate concentrations of DOC and sucrose to agar medium are useful for selective detection of aflatoxigenic fungi using the DV-AM method. Using this semi-selective medium and DV-AM method, some aflatoxigenic fungi were successfully isolated from some non-agricultural field in Fukui city, Japan. These conditions will be useful for screening of aflatoxigenic fungi from soils from various environments.A. parasiticus SYS-4 (NRRL2999) was used as an aflatoxigenic strain. A. parasiticus 95DM is a norsolorinic acid-accumulating mutant strain of the SYS-4 strain. A. parasiticus NIAH-26 is a mutant strain of the SYS-4, which produces neither AFs nor any pigmented AF precursors [A. nomius based on its calmodulin DNA sequence. Aflatoxigenic fungi isolated from soils in Fukui city were named using the prefix FUT-. FUT-A1 strain among them was used in this work. Rhizopus sp. was isolated from a soil in FUT, which was identified based on its morphological characters [ecursors . An aflaaracters .w/v) DOC-CP agar medium , 10% sucrose , 0.1% Na DOC , 0.1 g CP )/L, and 2% agar ) for the DV-AM method. The medium YES(2-10)-0.15% DOC-CP or YES(2-10)-0.05% DOC-CP medium was also used when specified.For the description of each medium, we named the concentrations of yeast extract and the carbon sources used by using a hyphen after the abbreviation of the medium. When necessary, a blanket together with the hyphen is also used to avoid confusion. We routinely used YES(2-10)-0.1% -CP agar medium supplemented with various concentrations of DOC, TX-100 ), or TW-80 ). Potato dextrose agar medium ) was used for morphological characterization. The GY(2-0.5) agar medium , 0.5% yeast extract, 2% agar) was used for the purification of each fungus.For the screening of the aflatoxigenic fungi from soils, 20 μL of 100-fold diluted DV (dichlorvos Fujifilm Wako)) methanol solution was spread onto YES(2-10)-0.1% DOC-CP agar medium to make YES(2-10)-0.1% DOC-CP-DV and then stored at 4 °C until usage.Rhizopus sp. was observed on the same plate, YES(2-10)-0.1% DOC-CP-DV medium was used for the second purification of the isolated fungus. The resulting fungal isolates were further purified by three repetitions of single conidiospore isolation.Each soil sample (approx. 0.15 g) was suspended in 0.5 mL of 0.05% TW-80 aqueous solution, and the resulting suspension (50 μL or 100 μL) was then spread onto YES-0.1% DOC-CP-DV agar plates or other plates. The plates were put upside-down and then cultured at 28 °C in darkness for 3 days. Each plate was treated with 200 μL of ammonium hydroxide solution  put in the inside of the lid of the petri plate. When color changes of colonies from pale yellow to red were observed from the underside of the plates, conidiospores of each red colony (candidate aflatoxigenic fungi) were immediately picked up with a sterile toothpick and inoculated onto a new GY(2-0.5) plate and cultured. When For the improvement of the culture media, a soil suspension (approx. 15 mg) of the FUT flowerbed was inoculated onto a YES(10-2)-CP-DV agar plate supplemented with various concentrations of DOC, TX-100, and TW-80 and then cultured at 28 °C for 3 days. Each conidiospore suspension (2 μL each) of the FUT-A1, SYS-4, and NIAH-26 strains was inoculated onto the same plate and then cultured for 3 days. The resulting plates were then treated with AM vapor, and color changes were then observed from the back side of the plate. The diameters of the resulting colonies were respectively measured.After the inoculation of fungi or soils, all petri dishes were shielded with Silkylite tape  and then cultured.Soil samples (approx. 10g) present up to 5 cm in depth were routinely collected into plastic bags from three sites in Fukui city, Japan: a flowerbed on the FUT campus, a mountain road, and a park in August–October 2017, and were kept at 4 °C until use.For the determination of the appropriate depth for soil collection, soils in the flowerbed on the FUT campus were collected from each plate at five 2 cm depths from the surface.Regarding the storage conditions of the soils, we repeated three sessions of freezing at 80 °C followed by thawing at room temperature. The resulting soil at each step was analyzed by the DV-AM method.For the analysis of the morphology  of aflatoxigenic fungi in the FUT flowerbed soil, a soil suspension with 0.05% TW-80 aqueous solution from a FUT flowerbed was filtered through a 50 μm mesh sheet, and the flow sample was pooled. The residue on the filter was washed twice with the same volume of the solution (1st wash and 2nd wash). The residue remaining on the filter was suspended with the same volume of the solution. Each pooled fraction was inoculated onto a YES-0.1% DOC-CP-DV plate and analyzed by the DV-AM method.A SYS-4 spore suspension was mixed with a soil suspension containing 15 mg or 30 mg FUT soil, in which no aflatoxigenic fungi had been detected, in a total volume of 260 μL. The resulting solution (100 μL each) was respectively inoculated onto YES-0.1% DOC-CP-DV medium and then cultured for 3 days. After AM treatment, the number of SYS-4 red colonies on the plate was counted. The recovery rate of the colonies was calculated by comparison with those (approx. 50 conidiospores) in the absence of soil. The same experiment was done in duplicate using two different FUT soils.g for 2 min, the upper extract (10 μL) was analyzed by TLC using a silica gel plate  and a developing solution of toluene-ethyl acetate-acetic acid .To confirm the AF productivity of the FUT-A1 strain and other fungi isolated from soils of Fukui city, Japan, we cut out an aliquot of the fungal colony together with the agar underneath the colony on an agar plate and transferred it to a new 1.5 mL microtube. Metabolites in the samples were extracted with 0.3 mL of ethyl acetate followed by mixing with a vortex mixer. After centrifugation at 10,000× For the determination of the morphological characters, each fungus was inoculated on PDA medium and cultured for 4 days. The colony was observed using a VHX digital microscope equipped with a VH-Z25 lens .cmd) gene of FUT-A1 was amplified by PCR using the enzyme KOD-Plus  and CMDF1 and CMDR1 primers [cmd gene of the fungi obtained were deposited in the DDBJ/EMBL/GenBank nucleotide sequence database under accession no. LC431242.For the identification of each isolated fungus, we collected the fungal mycelia on the GY(2-0.5) agar plate medium by using a pair of forceps, and then disrupted them with zirconium beads using Tris-EDTA-saturated phenol with FastPrep FP100A . After centrifugation, the upper aqueous layer containing DNA was diluted with sterile water and then applied for PCR. The calmodulin ( primers . The res


N = 48) and to parents of normally developing children (control group) (N = 87), and the intake of dietary omega-3 LC-PUFA, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), was estimated. Children with ADHD consumed fatty fish, lean fish, mollusks, crustaceans, and chicken eggs significantly less often (p < 0.05) than children in the control group. The estimated daily omega-3 LC-PUFA intake (EPA + DHA) was significantly below that recommended by the public health agencies in both groups, and was significantly lower in children with ADHD  compared to normally developing children. Dietary intervention to increase the consumption of fish and seafood is strongly advised and it is especially warranted in children with ADHD, since it could contribute to improve the symptoms of ADHD.Omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) play a central role in neuronal growth and in the development of the human brain, and a deficiency of these substances has been reported in children with attention deficit hyperactive disorder (ADHD). In this regard, supplementation with omega-3 polyunsaturated fatty acids is used as adjuvant therapy in ADHD. Seafood, particularly fish, and some types of nuts are the main dietary sources of such fatty acids in the Spanish diet. In order to assess the effect of the intake of common foods containing high amounts of omega-3 polyunsaturated fatty acids, a food frequency questionnaire was administered to parents of children with ADHD ( There is a growing evidence that several mental disorders, although they show an underlying genetic predisposition , are proPrevious studies refer to the fundamental role afforded by omega-3 LC-PUFA in several essential metabolic functions, given their implication in diverse neuronal processes, as well as in cell growth, the function of cell membrane, hormonal, and immunological cross-talk, and gene expression regulation ,22,23. A1)Evaluation of the pattern of consumption of the main dietary sources of food containing omega-3 LC-PUFA in children with ADHD and in a control group.2)Estimation of the daily intake of omega-3 LC-PUFA (EPA + DHA) in the two groups.3)Evaluation of the influence of age, sex, and body mass index (BMI) upon omega-3 LC-PUFA intake.Although many studies on omega-3 LC-PUFA supplementation in ADHD have been published in recent years ,14, mostAn observational case-control study was carried out in Valencia (Spain) in 2016–2017. The study participants were recruited among patients (children and adolescents) with ADHD undergoing child psychiatrist consultation. Neurologically healthy children (control group) were recruited from two public schools in Valencia (Spain). Attention deficit hyperactive disorder was confirmed based on the DSM-IV diagnostic criteria using a standard neurodevelopment examination and interview . The parents of children with ADHD were interviewed during ordinary consultation with the child psychiatrist. Clinical information  was retrieved by reviewing the medical records in the psychiatrist consultation of children with ADHD. Body mass index was calculated as weight in kilograms divided by the square of height in meters. For children and adolescents, BMI is age- and sex-specific, and is often referred to as BMI-for-age. According to the international guidelines, BMI was grouped into four categories: underweight (BMI less than the 5th percentile), normal or healthy weight (5th percentile to less than the 85th percentile), overweight (85th percentile to less than the 95th percentile), or obese  .The chilThe study protocol was approved by the local Ethics Committee of the University of Valencia  (protocol number H1397475950160). Parents signed the informed consent in order to participate in the study.The parents completed the food frequency questionnaire (FFQ) about their children’s diet and were also instructed to report all beverage and supplement consumption. The instrument was a semi-quantitative food questionnaire that was comprised of 136 food items, and is validated in Spain . SpecifiParents self-reported fish and nuts consumption in their children. Fish was defined as “any kind of fish, including fish sticks and canned tuna fish, shellfish, crustaceans and mollusks.” Participants reported: (a) how often they consumed fish ; and (b) the type of fish they typically consumed.The items of the three-day semi-quantitative food questionnaire  related n < 50) or Kolmogorov–Smirnoff (n ≥ 50) tests. As a result of non-normal data distribution, we used nonparametric tests, e.g., the Mann–Whitney U-test (when comparing quantitative variables between two groups) or the Kruskal–Wallis test (when comparing quantitative variables among three or more groups). Correlation analysis between quantitative variables was performed with the nonparametric Spearman test. In order to control the effect of intervening variables, partial correlations were performed. Differences between categorical variables were evaluated with the chi-squared test. In the case of food frequencies, we applied the z-test for differences between proportions to determine which of the five to seven categories differed between the control and ADHD groups. To quantify the effect size for two groups comparison we calculated Cohen’s d. Statistical significance was considered to be p < 0.05. The SPSS version 24.0 statistical package  was used throughout.In the univariate analysis, variables were represented as absolute frequencies and percentages for categorical variables, and as the mean ± standard deviation (SD) for continuous (quantitative) variables. In the bivariate analysis, we first checked for normal or non-normal data distribution for quantitative variables using the Shapiro–Wilk (p = 0.64) or mean age (p = 0.86). Regarding the weight distribution of the subjects, 18.5% (n = 25) of the sample had low weight (percentile < 5), 36.3% (n = 49) showed normal weight (percentile 5–84), 23.7% (n = 32) were overweight (percentile 85–94), and 21.5% (n = 29) were obese (≥95 percentile). Significant differences in weight distribution were observed between the control and ADHD groups (p < 0.0001). In relation to BMI, low weight was significantly more prevalent in the control group compared to the ADHD group (p < 0.0001), while obesity was significantly more frequent in the ADHD group compared to the control group (p < 0.001) (The characteristics of the study sample are shown in < 0.001) .p < 0.001) , “twice–four times a week”  and “five–six times a week” . Significant differences were also observed in the case of fatty fish   , “once–three times a month” , “once a week” , and “twice–four times a week” .The reported average energy intake was approximately 1705 kcal. Of this amount, 51% corresponded to carbohydrates, 34% to fat, and 15% to protein. Fish intake was significantly lower in children/adolescents with ADHD than among the controls for all types of fish and seafood, except codfish. Significant differences were recorded in relation to lean fish  (< 0.001) A; the z-ed test) B; the z-p < 0.001; chi-squared test); the z-scores analysis showed significant differences for the intake category “once a week” . The same applied to the intake of shellfish  (p < 0.05); the z-scores analysis showed significant differences for the intake category “twice–four times a week” . Likewise, significant differences were observed in the intake of mollusks  (p < 0.001); the z-scores analysis showed significant differences for the intake category “once a week” . Lastly, significant differences were recorded in the intake of crustaceans  ; the z-scores analysis showed significant differences for the intake categories “once a week”  and “five–six times a week” . In contrast, the intake of codfish was not significantly different between the two groups (p = 0.23).Significant differences were recorded in the intake of smoked fish  (p = 0.07), omega-3 LC-PUFA supplements (p = 0.26), or omega-3 fatty acid-enriched milk (p = 0.14). The intake of omega-3 fatty acids from nuts were not included in the calculation of daily EPA + DHA intake, since these foods contain other omega-3 LC-PUFA different from DHA and EPA, and because no significant differences in the intake of dry fruits were observed between the ADHD and control groups.There were no significant differences in relation to the consumption of nuts  (p < 0.05), and eggs , but not to other foods (p > 0.05).Significant differences in food intake were observed between females and males in relation to fatty fish and shellfish  (p < 0.05), fatty fish (p < 0.01), mollusks (p < 0.05), and other types of fish and seafood less frequently consumed (p < 0.05).The estimated ingestion of omega-3 LC-PUFA (EPA + DHA) in the diet was 109.87 ± 80.27 mg/day for the control group and 78.42 ± 56.64 mg/day for the children with ADHD ( = 0.45) . The anap < 0.05) and crustaceans . No significant differences were observed in the estimated daily amounts of omega-3 LC-PUFA (EPA + DHA) between sexes (p = 0.17) or among children in the different weight categories (p = 0.57).There was a significant correlation between the mean daily intake of EPA + DHA and the frequency of intake of fatty fish . The correlation between mean daily omega-3 LC-PUFA intake and age no longer proved significant  after controlling for the intervening variables, e.g., group, sex, and weight categories, suggesting that these contribute significantly to the association between omega-3 LC-PUFA intake and age.In contrast, a significant and direct correlation was observed between the intake of omega-3 LC-PUFA and of the age of the children . This finding agrees with those of a recent meta-analysis concluding that the prevalence of obesity in ADHD is 40% higher than in the general population ,52. The Our study has a number of limitations. First, the cross-sectional observational design involved limits regarding inferences about causality between insufficient intake of omega-3 LC-PUFA and the worsening of ADHD symptoms. Second, there were a number of issues related to the completion of records. Data referred to intake may contain errors due to inaccuracies in recorded quantities and they are based on parents’ reports rather than children’s. However, we are confident that the self-reported information provided by parents about the nutritional assessment of their children was adequate because they showed interest in the study and they received training and support in filling out the food records. Furthermore, the attrition rate was low. We, therefore, think that the study has a good internal validity. A third limitation is the fact that we did not measure the intake of omega-3 LC-PUFA coming from other sources. Nevertheless, we are confident about the main role of fish and seafood as the principal source of EPA + DHA in the Spanish diet ,39.In our sample of children with ADHD and controls (age- and sex-matched with the ADHD subjects), there were considerably more boys than girls , whicDespite these limitations, our study underscores the need for greater attention to the education of parents and children regarding healthy dietary habits in Spain, and as such, education is the most promising and practical complementary management strategy in ADHD. Given that fish consumption is the main source of dietary omega-3 LC-PUFA , interveThe intake of seafood in particular fish, is reduced in children with ADHD compared to typically developing children and this may contribute to reduced intake of some omega-3 LC-PUFA such as EPA and DHA, essential nutrients for a proper brain development and function. Further research is required to clarify associations between ADHD symptomatology, eating patterns and health status.


HLA-B*57:01 screening was added to clinical care guidelines in 2008 to reduce the risk of hypersensitivity reaction from abacavir. The uptake of HLA-B*57:01 screening and incidence of hypersensitivity reaction were assessed in a prospective clinical cohort in the United States to evaluate the effectiveness of this intervention.We included all patients initiating an abacavir-containing regimen for the first time in the pre-HLA-B*57:01 screening period  or the post-HLA-B*57:01 screening period . Yearly incidence of both HLA-B*57:01 screening and physician panel-adjudicated hypersensitivity reactions were calculated and compared.Of the 9619 patients eligible for the study, 33% initiated abacavir in the pre-screening period and 67% in the post-screening period. Incidence of HLA-B*57:01 screening prior to abacavir initiation increased from 43% in 2009 to 84% in 2015. The incidence of definite or probable hypersensitivity reactions decreased from 1.3% in the pre-screening period to 0.8% in 2009 and further to 0.2% in 2015 in the post-screening period.Frequency of HLA-B*57:01 screening increased steadily since its first inclusion in treatment guidelines in the United States. This increase in screening was accompanied by a decreasing incidence of definite or probable hypersensitivity reactions over the same period. However, a considerable proportion of patients initiating abacavir were not screened, representing a failed opportunity to prevent hypersensitivity reactions. Where HLA-B*57:01 screening is standard of care, patients should be confirmed negative for this allele before starting abacavir treatment. Abacavir, a nucleoside reverse transcriptase inhibitor (NRTI), was approved by the FDA in December 1998. It has since become widely used in combination with other antiretroviral agents to achieve viral suppression and immunologic improvement in patients with HIV infection . While aOver 90% of HSR occur in the first 6 weeks following abacavir initiation , 13. HypExcept for rare fatalities in cases of HSR among patients during their first exposure to abacavir, the symptoms are in general reversed after the discontinuation of abacavir. However, hypersensitivity reaction is much more severe and more likely to be fatal in patients who, after the resolution of initial symptoms, are reintroduced to abacavir. Additionally, there have been reports of individuals who were asymptomatic following initial abacavir use, but developed re-challenge hypersensitivity after use in a subsequent regimen , 19.A genetic link between the risk for abacavir HSR and specific human leukocyte antigen (HLA) alleles HLA-B*57:01 was identified, leading to the introduction of HLA-B*57:01 screening for clinical use in treatment guidelines in the United States on June 15, 2008 .The HLA-B*57:01 test was introduced and added to guidelines for clinical care over 10 years ago. The main objective of this study was to describe and compare the annual incidence rate of HLA-B*57:01 screening and HSR before and after June 15, 2008 to assess the use and effectiveness of screening on the occurrence of abacavir HSR in a real-world setting.®) cohort is a clinical cohort including patients from 79 HIV specialty outpatient clinics in 15 US states. For all patients receiving healthcare at a participating site; clinical diagnoses, medications prescribed, and laboratory results are captured prospectively through electronic medical records. Demographic and medical history information are also available. All data reflect routine medical care, with visits and testing scheduled at the discretion of the treating providers. Information captured in the electronic medical records system at each site is retrieved, cleaned, aggregated, and anonymized to maintain patient confidentiality. OPERA® complies with all HIPAA and HITECH requirements and received annual institutional review board approval by Advarra IRB including a waiver of informed consent and authorization for use of protected health information.The Observational Pharmaco-Epidemiology Research and Analysis  and proceeded until July 31, 2016. Study participants were included in the analysis population through January 1, 2016 to allow a minimum of 6 months of potential follow-up.The index date for an eligible patient was defined as the first date of the first abacavir-containing regimen ever prescribed to a patient after their first active visit in the OPERA database. Any abacavir-containing regimen was included in the analysis, regardless of formulation or combination with other antiretroviral drugs. The duration of the regimen was defined by discontinuation of abacavir. Changes to other medications within the ART regimen were not considered regimen changes if the patient remained on abacavir.Patients were observed from their index date until the first of the following censoring events: (i) discontinuation of abacavir, (ii) cessation of continuous clinical activity , (iii) death or (iv) study end .Time periods were defined based on the date of abacavir initiation (index date) as (i) pre-HLA-B*57:01 screening period, January 1, 1999 to June 14, 2008, and (ii) post-HLA-B*57:01 screening period, June 15, 2008 to July 31, 2016. Comparisons were made between pre- and post-HLA-B*57:01 screening periods.All instances of HLA-B*57:01 screening were captured prospectively through electronic medical records. Patients were categorized as screened on the date an HLA-B*57:01 screening was performed, regardless of the results.Patients with a diagnosis of HSR or symptoms potentially associated with HSR were identified for case adjudication by a panel of physicians. Relevant clinical information was abstracted from the electronic medical record for each of these patients, including symptomology, concurrent medications and diagnoses associated with these symptoms, as well as symptom progression.A panel of three physicians reviewed each patient’s anonymized clinical information independently to classify them as a definite, probable or possible case, or as not a case. Case determination was based on agreement between two or more of the three physicians. Disagreements on case classification were resolved by discussion among the physicians’ panel.A definite case of HSR was defined as one of the following within 6 weeks of abacavir initiation; (a) a diagnosis of “hypersensitivity reaction” or “allergic drug reaction” with specific reference to abacavir followed by drug discontinuation within 14 days from the time of onset, (b) two or more symptoms of a hypersensitivity reaction including abdominal pain, allergic reaction, cough, drug reaction, diarrhea, dyspnea, fatigue, fever, flushing, headache, hypersensitivity, malaise, nausea, pharyngitis, rash, or vomiting with complete remission within 14 days from abacavir discontinuation, or (c) a death within 14 days of a diagnosis or a symptom of HSR (cause of death data was not available). A probable case was defined as a single hypersensitivity symptom or other atypical complaints within 6 weeks of initiating abacavir which did not progressively worsen but did not remit until abacavir was discontinued. A possible case of HSR was defined as any of the above criteria greater than 6 weeks after abacavir initiation. Patients who subsequently tolerated abacavir re-challenge were deemed to not be a case.Data describing clinical and demographic patient characteristics were summarized using medians with interquartile ranges (IQR) for continuous variables and frequencies and proportions for categorical variables. Where applicable, statistical comparisons of patient characteristics by HLA-B*57:01 screening period were made using Pearson’s Chi square or Fisher exact tests for categorical variables and Wilcoxon rank-sum test for continuous variables.The incidence of HLA-B*57:01 screening and physician-adjudicated HSR was calculated for the pre- and post-HLA-B*57:01 screening periods. Temporal trends were assessed by plotting screening and HSR incidence over calendar years. Kaplan–Meier methods were used to assess time to HSR in the pre- and post-HLA-B*57:01 screening periods. Incidence density was described using incidence rates by year and compared over screening periods using Poisson regression to estimate the incidence rate ratio with 95% confidence intervals.Over time, physicians may have become more adept and confident at identifying HSR. The impact of the differential skill of the physician panel at identifying events in 2016, compared to individual physicians between 1999 and 2016 was therefore assessed. This sensitivity analysis included all diagnoses of HSR and all signs and symptoms consistent with HSR as recorded by the treating physician without regard to adjudication by the panel.Finally, the main analysis restricted the case definition to events occurring within 6 weeks of abacavir because over 90% of HSR occur within that time frame , 13 and Of the 15,648 HIV-1-positive patients who initiated their first abacavir-containing regimen between January 1, 1999 and January 1, 2016, 9619 were eligible for inclusion in the analysis population  patients were identified for review by a physician panel. Deaths within 14 days of an adjudicated HSR event were rare and did not differ between periods, with one death (0%) pre-screening and six deaths (0.1%) post-screening (p = 0.4370).Following adjudication, a definite, probable or possible HSR event occurred in 1.6% of patients in the pre-screening period and in 0.6% in the post-screening period (p < 0.0001). Definite, probable or possible HSR occurred a median of 20 days  after abacavir initiation in both periods (p = 0.9359).When only definite or probable adjudicated HSR were considered, the overall HSR incidence was further reduced to 1.3% pre-screening and 0.4% post-screening (p < 0.0001). The median time to definite or probable HSR was 17 days  in both periods (p = 0.7028).Incidence of definite or probable HSR decreased over the study period with a high of 1.8% in 2002 and a low of 0.2% in 2015  on abacavir, the overall HSR-free survival probability was 99.3%. The 6-weeks HSR-free survival was 98.7% in the pre-screening period and 99.6% in the post-screening period. The Kaplan–Meier plot for HSR survival revealed statistically significantly different curves by screening period (log-rank p < 0.0001)  compared to the pre-screening period, with 32 cases per 100,000 person-days  .Many additional HSR events were identified when all physician-adjudicated HSR were included in the second sensitivity analysis, regardless of proximity to abacavir use. Indeed, 22% of all definite or probable HSR identified occurred more than 6 weeks after abacavir initiation. When definite, probable or possible HSR were considered, 53% occurred after 6 weeks. Again, the overall incidence in the pre-screening period was elevated compared to the post-screening period for definite or probable HSR  and for definite, probable or possible HSR .® cohort, before the screening test was available, the overall definite or probable HSR incidence was 1.3%. HLA*B-57:01 screening increased steadily after its introduction in June 2008, from 43% screened in 2009 to 84% in 2015. This expansion of screening was accompanied by an important decrease of HSR incidence in the same period from 0.8% in 2009 to 0.2% in 2015.To the best of our knowledge, this is the first evaluation of HLA*B-57:01 screening practices and changes in HSR incidence in a clinical setting in the United States. In the OPERAEstimates of HSR incidence vary greatly based on the study population, clinical practices and case definition used. Clinically, suspected abacavir HSR is diagnosed and managed based on the onset of one symptom from two or more of the following categories: skin rash, fever, GI complaints , constitutional symptoms , or respiratory symptoms  .A review of clinical trials involving 5332 patients reported an overall HSR incidence of 3.7% . Among tThe 1.3% pre-screening period incidence of adjudicated HSR in this study is considerably smaller than the HSR incidences reported among trial participants. However, the incidence estimated in this large clinical cohort is larger than the estimate from the historic review of post-marketing spontaneous reports. These discrepancies can be explained in part by a closer monitoring during trials, and an underestimation of post-marketing events due to reliance on spontaneous reports. The use of electronic medical records in these OPERA analyses allowed a fuller representation of HSR incidence in routine care than spontaneous reports. This, combined with a thorough review of signs and symptoms by a panel of physicians, increased the validity of HSR adjudication and improved the accuracy of the estimates presented here.®, as epicutaneous patch testing for abacavir HSR is a research tool not widely used in clinical practice. Immunological confirmation of HSR greatly reduces its incidence, as demonstrated in a randomized controlled trial assigning patients to either prospective HLA*B-57:01 screening or immediate abacavir initiation with HLA*B-57:01 screening at the end of the 6-week follow-up. Among prospectively screened patients, 3.4% received a clinical HSR diagnosis, although none of the clinical cases were positive with epicutaneous patch testing. Among patients initiating abacavir without undergoing screening, 7.8% were clinically diagnosed, but only 2.7% were immunologically confirmed by epicutaneous patch testing [While physician panel adjudication of HSR improved the case definition in this study, immunological confirmation of HSR was not available in OPERA testing .Although most clinical cohort studies report a higher HSR incidence, a similar trend of considerable reductions in HSR incidence following the introduction of HLA*B-57:01 screening has been previously described in other settings. In Western Australia, HSR incidence dropped from 8% in the pre-screening period (1998 to 2001) to 2% in the post-screening period  . In the In our analysis, the incidence rate of definite or probable HSR was significantly lower in the post-screening period compared to the pre-screening period, with an incidence rate ratio of 0.31 . PREDICT-1, a randomized controlled trial reported a similar reduction in clinical HSR diagnoses when patients were prospectively screened rather than initiating abacavir immediately  .In this study, the median time to definite or probable HSR (defined as an event occurring within 6 weeks of abacavir initiation) was 17 days, which is longer than the median 8 to 11 days reported in trials , 13. A c® database, it was impossible to assess HSR-specific mortality. However, death within 14 days following HSR was very rare in this study (0.07%), and consistent with reports of 0.03% mortality in patients who received abacavir in clinical trials [Patients initiating abacavir in the post-screening period were significantly different from those initiating in the pre-screening period. These differences were consistent with the changing HIV epidemic over these time periods, notably as earlier initiation of effective ART results in better control of the infection  and longl trials .® database, the largest continuously operating cohort of HIV-infected patients, which includes complete patient health records managed in electronic health record systems and is updated daily. The HIV-infected population in OPERA® receives care at 79 separate locations throughout the United States and represents 7% of all HIV-infected patients linked to care in the United States. Therefore, the incidences of HLA*B-57:01 screening and HSR incidence estimates in this cohort are largely representative of changes in clinical practice in the United States.This study made use of the OPERAHowever, this study was restricted to patients initiating abacavir. Therefore, HLA*B-57:01 screening among patients who did not initiate abacavir was not assessed, resulting in an underestimation of true HLA*B-57:01 screening uptake. In addition, the HSR adjudication criteria used by the physicians’ panel was different from criteria used in other studies and the current approved product labelling . The ret® cohort. However, 16% of patients initiating abacavir in 2015 had not been screened beforehand. This gap indicates a potential for further reducing the risk of HSR by improving clinical practices. Indeed, where HLA-B*57:01 screening is standard of care, patients should be confirmed negative for this allele before starting abacavir treatment, as recommended in clinical guidelines.Expanded HLA*B-57:01 screening in clinical practice has been associated with fewer patients experiencing abacavir-associated hypersensitivity reactions in the OPERA


The underlying mechanisms of Parkinson´s disease are not completely revealed. Especially, early diagnostic biomarkers are lacking. To characterize early pathophysiological events, research is focusing on metabolomics. In this case-control study we investigated the metabolic profile of 31 Parkinson´s disease-patients in comparison to 95 neurologically healthy controls. The investigation of metabolites in CSF was performed by a 12 Tesla SolariX Fourier transform-ion cyclotron resonance-mass spectrometer (FT-ICR-MS). Multivariate statistical analysis sorted the most important biomarkers in relation to their ability to differentiate Parkinson versus control. The affected metabolites, their connection and their conversion pathways are described by means of network analysis. The metabolic profiling by FT-ICR-MS in CSF yielded in a good group separation, giving insights into the disease mechanisms. A total number of 243 metabolites showed an affected intensity in Parkinson´s disease, whereas 15 of these metabolites seem to be the main biological contributors. The network analysis showed a connection to the tricarboxylic cycle (TCA cycle) and therefore to mitochondrial dysfunction and increased oxidative stress within mitochondria. The metabolomic analysis of CSF in Parkinson´s disease showed an association to pathways which are involved in lipid/ fatty acid metabolism, energy metabolism, glutathione metabolism and mitochondrial dysfunction. Parkinson´s disease (PD) is a severe neurodegenerative disease with a prevalence of 0.6% in 65 to 69 years old population that increases up to 3.5% in the population between 85 and 89 years old . The cliet al. applied ultrahigh performance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry for the analysis of plasma and cerebrospinal fluid (CSF) metabolome of 49 PD affected patients [LeWitt patients . The idepatients . The moupatients , 8, a MSpatients , 11 to epatients , 8. A repatients .Generally, the metabolomics’ investigations can be targeted or non-targeted. A targeted investigation is restricted to a specific compound or a class of compounds whereas non-targeted analysis provides a comprehensive chemical characterization of a complex bio/chemical system . The anaA machine learning approach sorted the most important compounds in relation to their ability to differentiate PD and control. An additional network analysis revealed mass differences of the selected, most important compounds for supplemental information about ongoing processes. Notably, the network analysis showed a connection to the tricarboxylic acid (TCA) cycle, to mitochondrial dysfunction, and increased oxidative stress within mitochondria. With this unique combination of metabolic profiling and network analysis, the investigations provided deeper insights into ongoing disease mechanisms and the major effectors of the disease. Especially oxidative stress (e.g. lipid peroxidation and changes in antioxidant compounds like glutathione) and neuroinflammation (e.g. arachidonic acid) are known to be involved in PD  and hencThe purchased chemicals are: MeOH from CHROMASOLV LC-MS  and L-Arginine from Sigma Aldrich .A total of 126 CSF-samples were taken by standardized lumbar puncture at the Cologne University Hospital and were originally not intended for scientific use, but stored in the biobank of the hospital. The procedure of lumbar puncture was performed without problems and patients recovered quickly. Thirty-one of the CSF-samples were taken from patients diagnosed with PD and 95 samples derived from neurologically healthy controls . The conAfter lumbar puncture the samples remained at room temperature up to 6 hours for routine diagnostics. Subsequently, the samples were stored at -20±1°C temporary and later at -80±1°C until measurement. The count of erythrocytes was determined in a semi-quantitative manner in a counting chamber . Only samples with negative or isolated erythrocytes were involved in the study. The study was approved by the Ethics Committee of the University Cologne . All patients consented to scientific use of their CSF-samples.Prior to FT-ICR-MS analyses a protein precipitation extraction (PPE) was performed. The protocol was adapted from Forcisi et al. . The froUltrahigh resolution mass spectra were acquired by means of FT-ICR-MS , equipped with a 12 Tesla superconducting magnet  and an electrospray ionization (ESI) source . An external calibration was performed by analysis of a 3 mg/L arginine solution in MeOH with calibration errors below 0.1 ppm. All measurements were performed in negative ionization mode and ion accumulation time of 300 ms for higher sensitivity. The injection flow rate was 2 uL/min for electrospray. Operating temperature was 180°C for rapid solvent evaporation inside the electrospray. The ESI nebulizer gas flow rate was 2 L/min and the dry gas flow rate 4 L/min. The spectra were recorded in a mass-to-charge-ratio (m/z) range of 123–1000. For the generation of each mass spectrum 300 scans were acquired. A time-domain transient of 4 MW size was produced for each acquisition, which yielded ultra-high resolution for all signals, which are of metabolomic interest.Netcalc to remove potential spectral noise and isotope peaks. This software assigns molecular formulas to the aligned m/z-peaks based on a mass difference between the detected features [Homo sapiens as reference organism. All annotations were stored in the original data matrix. The assignment of a molecular formula to each m/z values was performed by molecular formula propagation through mass difference networks (MDiN). Here, m/z features (nodes) were connected by mass differences (edges) with corresponding molecular formula labels (e.g. Δm/z = 14.01565 → ΔCH2) and random walks starting from known peaks updated the molecular formulas of yet unassigned m/z peaks. The network was optimized to correct conflicting relationships and to closely follow the intrinsic m/z-error distribution of a spectrum for which reason this so-called Netcalc-algorithm is considered as an unsupervised filter that reduces the data size and reveals an underlying biochemical network structure inside the data set [2 and Q2 values. Moreover, we provide the p-value for Analysis of Variance of Cross-Validation Estimators. Those elaborations were done in SIMCA 13.0.3.0 .The spectra were calibrated with an in-house calibration tool developed in Matlab . The main principle is based on estimating the most probable calibration curve given the density map describing the behavior of a mass accuracy along the considered mass range. The extracted peaks were aligned within a 1 ppm tolerance window and stored in a data matrix . The masfeatures . Moreovefeatures , 20 withdata set . In ordedata set . The algdata set . For theFor the most important metabolites we did an analysis of covariance (ANCOVA) testing the significance for the interactions of the factor (Parkinson vs. control) with age and also with gender. Then we calculated all the p-values (adjusted by Dunnett test) of the differences between Parkinson vs. control  controlled by gender  was performed following Moritz et al. . The liset al. . The netet al. , 25. Fis2Y(cum) = 0.98 and Q2(cum) = 0.53. The cross validation Anova gave a p-value < 0.0001). The OPLS-DA explained 32% of variance with the first two components (the sPLS-DA explained with the three main components 16% of variance). The values of the Balanced Error Rate (BER) for the sPLS-DA model are in the supplementary The metabolic profiles of 31 Parkinson-patients and 95 controls were analyzed by FT-ICR-MS. A features selection was performed after the annotation of elemental formulas of the respective m/z-values in the generated data matrix. The feature selection excluded a part of the data noise and the information not related to the study design (Parkinson vs. control). The subset of variables calculated with such algorithm was reduced to 243 m/z values. Consecutively, we wanted to test if the list of 243 m/z was capable to separate the two groups under investigation. We built a sPLS-DA analysis  and an OFrom the subset of metabolites we investigated only the possible assigned with the KEGG database . Among those 32 candidates, we presented in Moreover, an ANCOVA analysis was performed to evaluate the influence of age and gender on the results. The analysis did not reveal any significant interactions between age and the main factor (Parkinson vs. control) for each of the metabolites presented in Based on the feature selection and the classification models results, the entire dataset  was analIn this study, we investigated the metabolic profile of CSF-samples from PD patients and controls. Our approach used CSF-samples to investigate the changes due to disease. CSF is a suitable biofluids to investigate changes in neurodegenerative diseases, because it is in close contact to the brain. This biofluids is in direct contact to the extracellular space of brain parenchyma  and therAs hypothesized, we revealed several altered metabolites in PD as compared to controls. Specifically, metabolites belonging to the lipid/ fatty acid, glutathione, and energy metabolism showed a strong shift. Especially the increased level of the fatty acid arachidonic acid is associated with increased oxidative stress and neuroinflammation –30.Age and gender are important factors in metabolic balance –33. The Statistical models were applied to the achieved mass spectra for calibration. All the models gave an overall agreement, isolating a common list of important masses altered in the two different groups. The statistical analysis models sPLS-DA and OPLS-DA confirmed the good group separation according to principal components 1 and 2. Moreover, the area under the curve (ROC) showed also a high performance of the classification model, since it was calculated based on the predicted scores . Additio2O2 [5-Oxoproline is an oxidation product and therefore elevated levels are a sign of increased oxidation. It is also associated with oxidative stress . 5-Oxopr2O2 . A highe2O2  and in n2O2 –40 and C2O2  of PD-pa2O2 , 42. Inc2O2 . Additio2O2  found anMetabolites belonging to the energy metabolism were found within our study. We discovered increased intensities of D-glucose-6-sulfate and α-mannosylglycerate and decreased intensity of sedoheptulose in CSF of PD-patients. The metabolite α-mannosylglycerate is part of the fructose and mannose metabolism, two compounds which were also found to be increased in CSF of PD-patients . These mal. found decreasing C16 and C18 fatty acids in plasma of PD patients [al. decreased amount of octenoic acid in serum [Our investigations showed several fatty acids to be altered in PD. Quinic acid, decanoic acid, 10-hydroxydecanoic acid, valerenic acid, arachidonic acid, and dihomo-γ-linolenic acid were found with increased intensities in PD. The lipid metabolism compounds DG, PC, and PE had decreased intensities in PD. Medium and long chain fatty acids  were also found increased in biofluids  of PD-patients by use of non-targeted metabolomic approaches . Other spatients  and Michin serum  indicati1 and PGE2 (anti-inflammatory/ pro-inflammatory) after dietary DGLA intake in rat plasma [1-level and the PGE2-level increased after DGLA-administration, but PGE1 increase was much higher. This finding is linked to increased anti-inflammatory capacity, at least at the beginning of disease. The ARA metabolism is strictly regulated in normal brain, but any misbalance due to neuroinflammation or oxidative stress can increase ARA metabolism in the brain and can finally cause neurodegeneration [et al. [2 was found in cortex and putamen in affected rats, which is associated with elevated neuroinflammation in the brains of diseased subjects. A disease-orientated investigation studied the metabolic changes in rat brain after an intravenous Mn-injection. K. Neth et al. [1, 15-(S)-HETE and Resolvin D2, which are associated with inflammation. Our investigation showed also a significant increase of DGLA and ARA in PD  were investigated several times in various body fluids. Both compounds are polyunsaturated fatty acids (PUFA) present in human brain. PUFAs are vulnerable to oxidative stress due to lipid peroxidation , 50. DGLt plasma . The PGEneration . Julien  [et al.  investig [et al. . Therebyh et al.  found deRA in PD , which cAdditional information about reactions was provided by MDEA. The MDEA analysis showed reactions with malondialdehyde (MDA)-production over-represented in PD. A direct detection of MDA by FT-ICR-MS is impossible because of too small molecular weight and the labile character of the compound. MDA is a marker for oxidative stress and a break-down product of PUFAs. Therefore, MDA is also involved in lipid peroxidation. Several studies identified MDA as marker of PD . They foMitochondrial dysfunction is known to be involved in PD , 59. MitIn conclusion, the pipeline we used gave us an understanding of the unknown space investigated. We found several metabolites in CSF of PD and controls by using the untargeted metabolomics technique FT-ICR-MS. Due to the use of multivariate statistical analysis a differentiation between PD and controls was possible. Especially metabolites of the lipid metabolism showed up to be affected due to disease. Moreover, indices of mitochondrial dysfunction and alterations of the energy metabolism were found in PD. More research effort should be directed to targeted approaches to unravel the lipid metabolism pathways affected in PD. Additionally, correlation of metal-analysis with TCA cycle products may enable further insights into disease mechanisms.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.


Surface plasmon enhanced light scattering (SP-LS) is a powerful new sensing SPR modality that yields excellent sensitivity in sandwich immunoassay using spherical gold nanoparticle (AuNP) tags. Towards further improving the performance of SP-LS, we systematically investigated the AuNP size effect. Simulation results indicated an AuNP size-dependent scattered power, and predicted the optimized AuNPs sizes  that afford extremely high signal enhancement in SP-LS. The maximum scattered power from a 130 nm AuNP is about 1700-fold higher than that obtained from a 17 nm AuNP. Experimentally, a bio-conjugation protocol was developed by coating the AuNPs with mixture of low and high molecular weight PEG molecules. Optimal IgG antibody bioconjugation conditions were identified using physicochemical characterization and a model dot-blot assay. Aggregation prevented the use of the larger AuNPs in SP-LS experiments. As predicted by simulation, AuNPs with diameters of 50 and 64 nm yielded significantly higher SP-LS signal enhancement in comparison to the smaller particles. Finally, we demonstrated the feasibility of a two-step SP-LS protocol based on a gold enhancement step, aimed at enlarging 36 nm AuNPs tags. This study provides a blue-print for the further development of SP-LS biosensing and its translation in the bioanalytical field. Surface plasmon resonance (SPR) has matured into one of the most powerful and versatile bioanalytical techniques. It has been widely employed to monitor biomolecular binding events including cells ,2,3, proWe have previously reported the novel concept of surface plasmon enhanced light scattering (SP-LS) biosensing . In this4·3H2O), sodium citrate, hydroquinone, Tween 20, N-hydroxy-succinimide (NHS), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), sodium chloride, hydroquinone and phosphate buffer saline (PBS) were purchased from Sigma Aldrich . HS-(PEG)7-COOH (MW 456.8 Da) was acquired from Polypure  and HS-(PEG)x-OMe (MW 2000 and 5000 Da) was obtained from Rapp Polymere . Mouse anti-goat IgG antibody (cat. No#. SAB3700264-1MG), Goat anti-mouse IgG antibody (cat. No#. SAB3701071-2MG), bovine serum albumin (BSA), horseradish peroxidase (HRP) enzyme were purchased from Sigma-Aldrich. Cellulose nitrate strip (pore size 0.45 µm) was purchased from Sartorius Stedim Biotech GmbH . PBST was prepared by addition of 0.1% tween-20 (Sigma-Aldrich) in PBS. TMB and H2O2 substrate were supplied by Zhengzhou Humanwell Biocell Biotechnology .Chloroauric acid (HAuCl4 (1%), sodium citrate (1%) and hydroquinone (0.03 M) at room temperature. The size distribution of the AuNPs was measured using a Zetasizer Nano ZS equipped with a 633 nm He-Ne laser from Malvern Instruments . Fluctuations in the intensity of scattered light (at 90° to the incident) were analyzed through the use of first-order and second-order autocorrelation functions. The Z-Average size and polydispersity index were obtained using the manufacturer’s software based on the cumulants method. UV-Vis absorption spectra were acquired on a Cary 5 UV-Vis-NIR spectrophotometer .Au seeds of ~17 nm size were synthesized by the standard citrate reduction method . AuNPs o4 with NH2OH at the volume ratio 6 to 1. In the solution phase, 1 volume of the as-prepared AuNPs was mixed with 1 volume of the GE reagent and the mixture was incubated at room temperature at three different time intervals. UV-Vis was used for monitoring the localized SPR (LSPR) shift of the AuNPs before and after the growth reaction. The size and morphology of the enlarged AuNPs were studied by SEM. In addition, the growth reactions were performed on the surface of SPR chip by exposing the chip surface-bound AuNPs to the GE reagent for 5 min.As-prepared AuNPs were enlarged following a gold enhancement (GE) protocol . The grov/v) ratio of HS-(PEG)7-COOH (2.18 mM) to HS-(PEG)x-OMe (2.18 mM) was added to the washed AuNPs and the mixture was incubated overnight at room temperature. The resulting PEGylated AuNPs were washed twice by centrifugation at 16,000 rpm for 10 min. The second pellet was reacted with a fresh solution of EDC/NHS (0.4 M/0.1 M) for 10 min at room temperature to activate the carboxylic groups of the PEGylated AuNPs. The excess EDC/NHS was washed off by centrifugation. Immediately, mouse anti-goat IgG (1 mg/mL) was incubated with the activated AuNPs for 3 h at room temperature. The excess reagents were removed from the bio-conjugated AuNPs by centrifugation of the mixture at 4 °C, and the samples were resuspended in PBST containing 1% BSA and stored at 4 °C until use.As-synthesized AuNPs were functionalized with goat anti-mouse IgG using EDC/NHS chemistry. Briefly, AuNPs were washed by centrifugation at 16,000 rpm for 10 min. A fresh 4:1  in PBS were spotted on cellulose nitrate strips. Goat anti-mouse IgG (1 mg/mL) was spotted as control. The blotted samples were left for 15 min at room temperature to air-dry. Then, the strip was immersed in 5% BSA for 30 min at room temperature as a blocking step. The strip was incubated with the antibody-AuNP (17 nm) conjugate (OD = 0.2) for 2 h at room temperature. The excess AuNPs were washed off twice with MQ water containing 0.05% Tween-20. The color change was observed by the naked eyes.48 nm of gold film on top of 2 nm of Cr film was sputtered on LaSFN9 glass to prepare SPR sensors as previously described . The deti under the illumination of a 632.8 nm excitation light, and 0.6 + 2.25i for the AuNPs. The refractive index for the chromium (Cr) film is 3.14 + 3.31i, and it is 1.333, 1.845, and 1.46 for water, prism LaSFN9, and the dielectric coating, respectively.The simulation of the SPR-LSPR coupling system was conducted with the three-dimensional (3D) finite element method using COMSOL Multiphysics 5.3 with the RF Module. The computational domain is a unit cell consisting of one AuNP surrounded by water sitting on a multilayered SPR substrate containing a prism LaSFN9, 2 nm-thick Cr film, 48 nm-thick Au film, and a 10 nm-thick dielectric coating. The 10 nm spacing was chosen based on the approximated distance between the Au film and AuNP tags for the anti-IgG model system used in the experimental study. The lateral dimension of the unit cell was set as the pitch p, i.e., the center-to-center distance between AuNPs. Periodic boundary conditions were employed at the sides of the unit cell, while water and LaSFN9 perfectly matched layers were applied at the top and bottom of the analyzed structure. In the simulations, the refractive index of the 48 nm-thick Au film was taken as 0.18 + 3.5A TM-polarized excitation light source with the fixed wavelength of 632.8 nm was introduced in the LaSFN9 domain. The incident light propagates from the prism LaSFN9 into the Au film-AuNPs coupling system, and was absorbed, reflected, or transmitted on striking the coupling system. The absorbed power was calculated by the volume integration of the resistive heating in the metallic nanostructure . The reflected (or transmitted) power was calculated by the surface integration of far-field power flow in the prism LaSFN9 (or water) domain. The incident angle of the TM-polarized light was varied to match the SPR condition.After solving the 3D Maxwell’s equations, the absorption, reflection, and transmission as a function of the incident angle was obtained. The scattered power from AuNPs was computed through the difference of the transmitted power with and without AuNPs, and normalizing it with the area of the simulated unit cell. In addition, the electric field distribution at the resonant angle was obtained from the COMSOL simulations.In the SP-LS sensing mode, SPs are excited with the Kretchmann configuration , when thcase see b. The insca for isolated AuNPs with different sizes were compared in The AuNP size-dependent scattered power as a function of the incident angle is shown in Simulation demonstrated that large AuNPs are preferable as they induce greater scattered signals. AuNPs with various sizes were therefore synthesized to experimentally investigate the AuNP size-dependence in the SP-LS scheme. AuNPs were first PEGylated prior to the bioconjugation of antibodies. However, significant aggregation occurred for larger AuNPs (> 64 nm) during the antibody modification step, which prevented their utilization in SP-LS measurements. The optimization of PEG coatings is not trivial as the polymeric biointerface plays a crucial role in both maintaining the colloidal stability of the samples as well as controlling the immuno-binding efficiency to biological targets ,30. The An IgG immuno-dot blot assay was employed to further confirm antibody functionality after conjugation onto AuNPs. As depicted in To further investigate the specificity and sensitivity of the biofunctionalized AuNPs in standard SPR refractometric biosensing, 17 nm AuNPs were PEGylated and conjugated with goat anti-mouse IgG antibody as described above. Mouse anti-goat IgG functionalized SPR sensors were exposed to goat anti-mouse IgG modified AuNPs of increasing concentrations and both the reflectivity versus angular shift and kinetic measurement were collected as shown in In our previous work, we have shown that 36 nm AuNPs used as SPR signal amplification tag yield approximately three orders of sensitivity enhancement in SP-LS sensing over conventional refractometric SPR. Simulation experiments shown in Further optimization of the bioconjugation strategy is warranted to fully take advantage of the huge signal increase in the SP-LS scheme for very large AuNPs. However, in order to harness the potential of large AuNPs as a signal amplification tag, we investigated the feasibility of a two-step process referred to as AuNP enlargement mediated SP-LS. Gold and silver enhancement procedures have been widely employed for the enlargement of AuNP tags in immunoassays. In this work, PEGylated AuNPs with diameters of 17 to 64 nm were first enlarged with GE reagents in solution and characterized by SEM to monitor the resulting size increase.As shown in Building on our previous report of the exquisite sensitivity afforded by SP-LS sensing scheme in sandwich immunoassay format, we have investigated the size-dependent effect of AuNP signal amplification tags in the SP-LS sensing scheme, from both theoretical and experimental standpoints. Simulation demonstrated the direct relationship between the NP size and scattered signals. AuNPs with sizes of 100–130 nm were predicted to induce extremely high scattered signal. We then experimentally optimized a coating procedure based on mixed PEG molecules to maximize biding to solid substrate, while preventing aggregation of the AuNPs. However, AuNPs larger than 64 nm could not be satisfactorily bioconjugated with monoclonal antibodies, preventing their application in the SP-LS experimental study. Using a model immunoassay, we confirm the AuNP size-dependence of the SP-LS scheme. AuNPs with diameters of 50 nm and 64 nm provided a significantly increased signal in comparison to 17 and 36 nm AuNPs. Finally, in order to fully harness the potential of large AuNPs (>64 nm) as signal amplification tags for SP-LS, this study shows that a two-step protocol based on the gold enlargement of the AuNPs following their binding to their molecular targets on the sensor surface can be implemented.


AimTo study the tibial tuberosity-trochlear groove distance (TT-TG) in normal Indian population and the variation of the same in relation to tibial size using computed tomography (CT) of knee.MethodsCT of 100 knees  were assessed. TT-TG distance and maximal medio-lateral (MML) distance of tibia was measured on axial CT scans. The modified TT-TG (mTT-TG) was calculated as the ratio of TT-TG and MML.ResultsThe average TT-TG distance was 13.01 (±2.84) mm for the entire group with males and females having 12.82 (±2.95) and 13.32 (±2.66) mm, respectively (p > 0.05). The MML distance was 75.99 (±3.78) and 66.77 (±4.33) mm for males and females, respectively (p < 0.05). The average modified TT-TG was 0.18 ± 0.04. The TT-TG distance of Indian knees was similar to values obtained in Caucasian knees and higher than other Asian knees (p < 0.05).ConclusionThe average TT-TG distance in Indian population is 13.01 mm, with no difference between males and females. The ML/TT-TG ratio was 0.18. The TT-TG distance in Indian population is found to be similar to the Western population and significantly higher than other Asian population. Tibial tuberosity-trochlear groove distance (TT-TG) relates to the relationship of the tibial tuberosity with the femoral trochlea and is used to assess the degree of external torsion and lateralisation of the tibial tuberosity in patellar instability . UnderstWe here present a CT-based assessment of TT-TG distance in normal Indian population and the variation of the same in relation to tibial size. The null hypothesis kept at the start of the study was that TT-TG distance in Indian population was different from Western population.This was a CT scan-based observational study of 100 knees. CT scans of patients who had undergone CT angiography of lower limbs for various reasons with normal knee CT, were used for the study. The sample size of 100 knees was calculated on the basis of study by Mullaji et al. . CT scanThe TT-TG distance was measured between the most anterior point of tibial tuberosity with the deepest bony point of trochlear groove which is perpendicular to the tangent to bony borders of the posterior condyles of distal femur on axial CT scans . First, Measurements were made using Horos Dicom viewer. The data obtained was assessed using Microsoft Excel 2010 and XL-STAT software. The values were expressed as mean ± standard deviation. The normality of test was assessed with Shapiro-Wilk test. Student’s t test or Wilcoxon-Mann-Whitney test was used to assess the statistical significance and p-value of <0.05 was considered significant. The normal range of MML-TT-TG was determined with 95% confidence interval. The TT-TG values obtained in our study were compared with similar studies in literature on Caucasian and other Asian population ,9. T-tesThe mean TT-TG distance for the entire study group was 13.01 mm (±2.84). There was no significant difference in TT-TG distance between male and female population (p > 0.05). The MML distance was 75.99 mm (±3.78) and 66.77 mm (±4.33) for the male and female group, respectively. This was found to be statistically significant (p < 0.05). The average ML/TT-TG ratio (mTT-TG) was 0.18 (±0.04). There was significant difference in this ratio between male and female subjects. The ML/TT-TG ratio averaged 0.18 (±0.04) with males and females having 0.17 (±0.04) and 0.2 (±0.05), respectively. This difference was found to be statistically significant (p < 0.05)  mm for Korean population . There wPatellar instability is a relatively common orthopaedic problem for which the TT-TG distance is a predominant factor in evaluation. An increased tibial tuberosity-trochlear groove distance is considered as an isolated and independent predisposing factor leading to patellofemoral instability and even more as an indication for performing distal realignment procedure around knee like medialisation of the tibial tuberosity . It is oThe tibial tuberosity position relative to the femoral trochlea is pertinent for the quadriceps mechanism to function normally as it decides the direction of the inferolateral force vector on the patella and pull of the quadriceps mechanism . In a noA TT-TG distance in the range 10-15 mm is usually considered as normal, and an increased distance is an indication for surgical intervention in the form of medial tibial tuberosity transfer to correct patellofemoral malalignment . HoweverOur study revealed the mean TT-TG distance to be 13.01 ±2.84) mm with no significant difference between gender. Twenty-four percent of the subjects had a value of more than 15 mm. Dejour et al. used CT scans with improved accuracy and reliability to calculate the TT-TG distance [4 mm withPennock et al. found an association of TT-TG distance with height and observed a variation of TT-TG distance by 0.12 with each centimetre increment . BalcareThe study has its limitations. Firstly, the sample size we had in our study was small. But, this is comparable to the rest of the literature at present. Secondly, MRI-based assessment of the TT-TG distance is being considered by many as a single investigation that would identify the underlying pathology and assess the status of the cartilage. But, there is a lack of clarity in respect to the validity of the techniques as Aarvold et al. have demonstrated that the MRI done with dedicated knee coils often gives altered distance . This stThe present study reveals the average TT-TG distance to be 13.01 mm in Indian population with males having average distance of 12.82 mm and females 13.32 mm. The ML/TT-TG ratio was 0.18. The TT-TG distance in Indian population is found to be similar to the Western population and significantly higher than other Asian population.